A detergent composition comprising:

The present invention relates to a laundry detergent composition which may for example, be formulated as a particulate, tablet or liquid composition.
Conventional laundry detergent powders intended for the handwash contain a substantial level of anionic surfactant, most usually alkylbenzene sulphonate. Anionic surfactants are ideally suited to the handwash because they combine excellent detergency on a wide range of soils with high foaming. However, event at high levels of anionic surfactant, for handwashing, the oily soil removal performance of such products is in need of improvement.
GB-A-1 570 128 discloses detergent compositions comprising from 4 to 40 wt % of a magnesium-insensitive surfactant (for example, alkyl ether sulphate, ethoxylated nonionic surfactant, amine oxide), from 13 to 40 wt % of sodium silicate, and from 5 to 50 wt % of a magnesium-selective detergency builder (for example, zeolite, sodium citrate, nitrilotriacetate, or calcite/carbonate).
JP-A-09 087 690 discloses a high-bulk-density granular detergent composition for machine wash use, containing anionic (5 to 40 wt %), plus ethoxylated nonionic surfactant (1 to 15 wt %), zeolite (10 to 40 wt %), and crystalline and amorphous sodium silicates (0.5 to 10 wt %). The anionic surfactant optionally contains alpha-olefin sulphonate (up to 10 wt % in the examples).
WO-A-97/43366 relates to detergent composition comprising from 0.5% to 60% by weight an anionic surfactant and from 0.01% to 30% by weight of a cationic surfactant. In the exemplified compositions in this document, the level of anionic surfactant is typically from about 10% to 15% by weight of the composition and the amount of cationic surfactant is from about 1% to 4% by weight. In some examples, the anionic surfactant component contains small amounts of an alkyl ether sulphate surfactant, typically at from 0.76% to 2.5% by weight of the total composition. Compositions with small amounts of alkyl ether sulphate anionic surfactant (up to xe2x88x921.75wt %) and in one case, a low amount of zeolite (1.5wt %) are disclosed in WO-A-98/01521. However, these compositions are spray-dried and therefore have high amounts of sodium sulphate which is a non-alkaline non-functional water-soluble salt (electrolyte).
Other spray-dried compositions with high levels of sodium sulphate and containing alpha-olefin sulphate anionic surfactant, together with 12 or 15 wt % zeolite builder are disclosed in JP-A-62 218499.
WO-A-00/40682 discloses compositions comprising more than 40% of an anionic surfactant system at least some comprising a calcium tolerant surfactant such as an alpha-olefin sulphonate or alkyl ether sulphate. The examples with calcium tolerant surfactant either contain no phosphate or aluminosilicate builder or else a very high level of phosphate.
Our UK Patent Application No. 9925961.6 also discloses compositions with 5-30 wt % of alpha-olefin sulphonate. These are all based on a calcite/carbonate builder system. Other compositions containing calcium tolerant surfactants are disclosed in U.S. Pat. No. 6,010,996, GB-A-2 309 706, U.S. Pat. No. 5,415,812, U.S. Pat. No. 4,265,777 and U.S. Pat. No. 4,970,017.
There remains a need, fulfilled by the present invention, for compositions which contain a strong builder, namely a phosphate or aluminosilicate builder, but at lower levels, yet which are still robust across a wide range of water hardness.
A detergent composition comprising:
(a) more than 10 wt % of a calcium-tolerant non-soap anionic surfactant system;
(b) from 0.1% to 10%, preferably from 0.5% to 10%, more preferably from 1% to 10% by weight of a strong builder system selected from phosphate builders and aluminosilicate builders and mixtures thereof; and
(c) the balance, if any, being other detergent ingredients wherein said composition comprises less than 35wt %, preferably less than 25 wt %, more preferably less than 15 wt % of non-functional non-alkaline water soluble inorganic salt.
The composition of the invention is based on mixed calcium tolerant anionic surfactant systems, plus moderate amounts of phosphate and/or aluminosilicate builder.
The composition of the invention contains more than 10 wt % of calcium tolerant anionic surfactant. Typically, the upper level of this calcium tolerant anionic surfactant system will be 75 wt %. However, preferably, the anionic surfactant system is present at from 15 wt % to 65 wt %, more preferably from 20 wt % to 60 wt %, especially from 30 wt % to 50 wt % based on the weight of the total composition.
The calcium ion stability of anionic surfactants can be measured by the modified Hart method (Witkes, et al. J. Ind. Encl. Chem. 29, 1234-1239 (1937)), carried in microtiter plates. The surfactant solution is titrated with a calcium ion solution. The onset of turbidity indicates the start of formation of insoluble calcium precipitates after a minute of shaking at room temperature.
As referred to herein, a xe2x80x9ccalcium tolerantxe2x80x9d surfactant is one that does not precipitate at a surfactant concentration of 0.4 g/L (and at a ionic strength of a 0.040 M 1:1 salt solution at) with a calcium concentration up to 20xc2x0 FH (French hardness degrees), i.e. 200 ppm calcium.
The preferred non-soap calcium tolerant anionic surfactant for use in the compositions of the present invention is alpha-olefin sulphonate.
Advantageously alkyl ether sulphate (another non-soap calcium tolerant material) may be present, preferably as a co-surfactant with the alpha-olefin sulphonate, in an amount less that that of the latter.
A preferred surfactant system comprises alpha-olefin sulphonate and alkyl ether sulphate in a weight ratio of from 5:1 to 15:1.
Other calcium-tolerant anionic surfactants that may be used alone or in combination with these or other calcium-tolerant anionics are alkyl ethoxy carboxylate surfactants (for example, Neodox (Trade Mark) ex Shell), fatty acid ester sulphonates (for example, FAES MC-48 and ML-40 ex Stepan), alkyl xylene sulphonates, dialkyl sulphosuccinates, fatty acid ester sulphonates, alkyl amide sulphates, sorpholipids, alkyl glycoside sulphates and alkali metal (e.g. sodium) salts of saturated or unsaturated fatty acids.
More than one other anionic surfactants may also be present. These may for example be selected from one or more of alkylbenzene sulphonates, primary and secondary alkyl sulphates.
When it is desired to avoid the calcium intolerance of alkylbenzene sulphonate surfactant altogether, then the anionic surfactant system may comprise (preferably at a level of 70 wt % or more of the total anionic surfactant) or consist only of one or more calcium-tolerant non-soap anionic surfactants.
The strong builder system is selected from phosphate builders, aluminosilicate builders and mixtures thereof. The total amount of this strong builder system is from 0.1% to 10%, preferably from 0.5% to 10%, more preferably from 1% to 10% by weight of the composition. However, one or more weak builders such as calcite/carbonate, citrate or polymer builders may also be present.
The phosphate builder (if present) may for example be selected from alkali metal, preferably sodium, pyrophosphate, orthophosphate and tripolyphosphate, and mixtures thereof.
The aluminosilicate may be, for example, selected from one or more crystalline and amorphous aluminosilicates, for example, zeolites as disclosed in GB 1 473 201 (Henkel), amorphous aluminosilicates as disclosed in GB 1 473 202 (Henkel) and mixed crystalline/amorphous aluminosilicates as disclosed in GB 1 470 250 (Procter and Gamble); and layered silicates as disclosed in EP 164 514B (Hoechst
The alkali metal aluminosilicate may be either crystalline or amorphous or mixtures thereof, having the general formula: 0.8-1.5 Na2O. Al2O3. 0.8-6 SiO2.
These materials contain some bound water and are required to have a calcium ion exchange capacity of at least 50 mg CaO/g. The preferred sodium aluminosilicates contain 1.5-3.5 SiO2 units (in the formula above). Both the amorphous and the crystalline materials can be prepared readily by reaction between sodium silicate and sodium aluminate, as amply described in the literature. Suitable crystalline sodium aluminosilicate ion-exchange detergency builders are described, for example, in GB 1 429 143 (Procter and Gamble). The preferred sodium aluminosilicates of this type are the well-known commercially available zeolites A and X, and mixtures thereof.
The zeolite may be the commercially available zeolite 4A now widely used in laundry detergent powders. However, according to a preferred embodiment of the invention, the zeolite builder incorporated in the compositions of the invention is maximum aluminium zeolite P (zeolite MAP) as described and claimed in EP 384 070A (Unilever). Zeolite MAP is defined as an alkali metal aluminosilicate of the zeolite P type having a silicon to aluminium ratio not exceeding 1.33, preferably within the range of from 0.90 to 1.33, and more preferably within the range of from 0.90 to 1.20.
Especially preferred is zeolite MAP having a silicon to aluminium ratio not exceeding 1.07, more preferably about 1.00. The calcium binding capacity of zeolite MAP is generally at least 150 mg CaO per g of anhydrous material.
The compositions of the invention must contain less than 35wt %, preferably less than 25wt %, more preferably less than 15wt % of non-functional non-alkaline water soluble inorganic salts such as water-soluble builders and alkaline agents other than alkali metal carbonates, silicates, metasilicates etc.
The compositions of the present invention optionally contain at least 0.1% by weight of the cationic surfactant system. Typically, the cationic surfactant system may be present at up to 15 wt % of the total composition. However, it preferably is present at from 0.5 wt % to 5 wt % at the composition.
Preferred cationic surfactant types include those having the formula:
R1R2R3R4N+Xxe2x88x92
wherein
R1=is a hydrophobic group (preferably) C12-14 alkyl, or derived from Coco;
R2, R3 or R4 are independently hydroxyethyl, hydroxypropyl or C1-4 alkyl (e.g. Me, Et) group;
Xxe2x88x92 is a solubilising cation, preferably Clxe2x80x94, Brxe2x80x94 or MeSO4xe2x88x92
Specific examples are:
The definition of cationic surfactants also include QMEA (quaternised monoethanolamine) or QTEA (quaternised triethanolamine). Quaternisation can arise as the result of neutralisation in situ of MEA or TEA by a surfactant acid (i.e. LAS acid, fatty acid, paraffin sulphonic acid =SAS acid, etc.):
The physical form of the amines is water-like liquid. They could be either applied in the slurry for powder making (as an alkali replacing caustic soda partly) or in a post-dosed granule adjunct and present as a counter-ion after the neutralisation of a surfactant acid.
(vi). APA Quats: Alkyl amido-N-propylene dimethyl (quarternary-)ammonium chloride (alkyl-CON(X)-(CH2)3NH+(CH3)2 Clxe2x80x94 where X=H or Me, Et, Pr or higher) APA Quats C12-C14-CON(X)(CH2)3 2 methyl
(vii) Alcohol triethoxy dimethyl ammonium chloride (alkyl (EO)n N+(CH3)3 Clxe2x80x94, where n is 1-10
(viii) Esterquats: Mono or di (cocoyl-ethylene) hydroxyethyl methylammonium chloride (Cocoyl(CH2)2N+(CH3)(CH2CH2OCH) CH3SO4xe2x88x92
(viii) Benzyldimethyl cocoalkyl ammonium chlorides (eg R(CH3)2N+CH2C6H5 Clxe2x80x94, etc.) ex AKZO-Nobel e.g. Arquad DMCB-80, DMHTB-75 Arquad M2HTB-80)
(ix) Cocobenzyl-(ethoxylated (2)-ammonium chloride (e.g. Ethoquad C12/B)
(x) Ethoxylated Quaternary Salts (Monoalkyl ethoxylates) i.e. monoalkyl methyl [ethoxylated (n)] ammonium chloride
e.g. Ethoquat 18/12, i.e. octadecylmethyl [ethoxylated (2)], Ethoquad 18/25, Ethoquad C/12, C/25, Ethoquad O/12, i.e. Oleylmethyl [ethoxylated (2)], ethoquad C/12 nitrate, i.e. cocomethyl [ethoxylated (2)] ammonium nitrate, Ethoquad T/12 i.e. Tallowalkylmethyl [ethoxylated (2)]-ammonium chloride.
If desired, nonionic surfactant may be included in order to control foam. The amount of these materials, in total, should not generally exceed 10 wt % and preferably will not exceed 5 wt %.
Preferred nonionic surfactants are the C10-C16 aliphatic alcohols having an average degree of ethoxylation of from 1 to 10, more preferably the C12-C15 alcohols having an average degree of ethoxylation of from 2 to 8.
Laundry wash compositions according to the invention may also suitably contain a bleach system. Fabric washing compositions may desirably contain peroxy bleach compounds, for example, inorganic persalts or organic peroxyacids, capable of yielding hydrogen peroxide in aqueous solution.
Suitable peroxy bleach compounds include organic peroxides such as urea peroxide, and inorganic persalts such as the alkali metal perborates, percarbonates, perphosphates, persilicates and persulphates. Preferred inorganic persalts are sodium perborate monohydrate and tetrahydrate, and sodium percarbonate.
Especially preferred is sodium percarbonate having a protective coating against destabilisation by moisture. Sodium percarbonate having a protective coating comprising sodium metaborate and sodium silicate is disclosed in GB-A-2 123 044.
The peroxy bleach compound is suitably present in an amount of from 0.1 to 35 wt %, preferably from 0.5 to 25 wt %. The peroxy bleach compound may be used in conjunction with a bleach activator (bleach precursor) to improve bleaching action at low wash temperatures. The bleach precursor is suitably present in an amount of from 0.1 to 8 wt %, preferably from 0.5 to 5 wt %.
Preferred bleach precursors are peroxycarboxylic acid precursors, more especially peracetic acid precursors and pernoanoic acid precursors. Especially preferred bleach precursors suitable for use in the present invention are N,N,Nxe2x80x2,Nxe2x80x2,-tetracetyl ethylenediamine (TAED) and sodium noanoyloxybenzene sulphonate (SNOBS). The novel quaternary ammonium and phosphonium bleach precursors disclosed in U.S. Pat No. 4,751,015 and U.S. Pat. No. 4,818,426 and EP-A402 971, and the cationic bleach precursors disclosed in EP-A-284 292 and EP-A-303 520 are also of interest.
The bleach system can be either supplemented with or replaced by a peroxyacid. examples of such peracids can be found in U.S. Pat. No. 4,686,063 and U.S. Pat. No. 5,397,501. A preferred example is the imido peroxycarboxylic class of peracids described in EP-A-325 288, EP-A-349 940, DE-A-382 3172 and EP-A-325 289. A particularly preferred example is phtalimido peroxy caproic acid (PAP). Such peracids are suitably present at 0.1-12%, preferably 0.5-10%.
A bleach stabiliser (transition metal sequestrant) may also be present. Suitable bleach stabilisers include ethylenediamine tetra-acetate (EDTA), the polyphosphonates such as Dequest (Trade Mark) and non-phosphate stabilisers such as EDDS (ethylene diamine di-succinic acid). These bleach stabilisers are also useful for stain removal especially in products containing low levels of bleaching species or no bleaching species.
An especially preferred bleach system comprises a peroxy bleach compound (preferably sodium percarbonate optionally together with a bleach activator), and a transition metal bleach catalyst as described and claimed in EP-A-458 397, EP-A-458 398 and EP-A-509 787.
Laundry wash compositions according to the invention may also contain one or more enzyme(s). Suitable enzymes include the proteases, amylases, cellulases, oxidases, peroxidases and lipases usable for incorporation in detergent compositions. Preferred proteolytic enzymes (proteases) are, catalytically active protein materials which degrade or alter protein types of stains when present as in fabric stains in a hydrolysis reaction. They may be of any suitable origin, such as vegetable, animal, bacterial or yeast origin.
Proteolytic enzymes or proteases of various qualities and origins and having activity in various pH ranges of from 4-12 are available and can be used in the instant invention. Examples of suitable proteolytic enzymes are the subtilisins which are obtained from particular strains of B. Subtilis B. licheniformis, such as the commercially available subtilisins Maxatase (Trade Mark), as supplied by Gist Brocades N.V., Delft, Holland, and Alcalase (Trade Mark), as supplied by Novo Industri A/S, Copenhagen, Denmark.
Particularly suitable is a protease obtained from a strain of Bacillus having maximum activity throughout the pH range of 8-12, being commercially available, e.g. from Novo Industri A/S under the registered trade-names Esperase (Trade Mark) and Savinase (Trade-Mark). The preparation of these and analogous enzymes is described in GB 1 243 785. Other commercial proteases are Kazusase (Trade Mark obtainable from Showa-Denko of Japan), Optimase (Trade Mark from Miles Kali-Chemie, Hannover, West Germany), and Superase (Trade Mark obtainable from Pfizer of U.S.A.). Detergency enzymes are commonly employed in granular form in amounts of from about 0.1 to about 3.0 wt %. However, any suitable physical form of enzyme may be used.
The compositions of the invention may contain alkali metal, preferably sodium carbonate, in order to increase detergency and ease processing. Sodium carbonate may suitably be present in amounts ranging from 1 to 60 wt %, preferably from 2 to 40 wt %. However, compositions containing little or no sodium carbonate are also within the scope of the invention.
Powder flow may be improved by the incorporation of a small amount of a powder structurant, for example, a fatty acid (or fatty acid soap), a sugar, an acrylate or acrylate/maleate copolymer, or sodium silicate. One preferred powder structurant is fatty acid soap, suitably present in an amount of from 1 to 5 wt %.
Yet other materials that may be present in detergent compositions of the invention include sodium silicate; antiredeposition agents such as cellulosic polymers; inorganic salts such as sodium sulphate; lather control agents or lather boosters as appropriate; dyes; coloured speckles; perfumes; foam controllers; fluorescers and decoupling polymers. This list is not intended to be exhaustive.
The compositions of the invention may be prepared by any suitable process.
The choice of processing route may be in part dictated by the stability or heat-sensitivity of the surfactants involved, and the form in which they are available.
For example, alpha-olefin sulphonate is robust, and is available in powder, paste and solution form.
Alkyl ether sulphate is more sensitive to heat, is susceptible to hydrolysis, and is available as concentrated (e.g. about 70% active matter) aqueous paste, and as more dilute (e.g. 28.5 wt %) solution.
In all cases, ingredients such as enzymes, bleach ingredients, sequestrants, polymers and perfumes which are traditionally added separately (e.g. enzymes postdosed as granules, perfumes sprayed on) may be added after the processing steps outlined below.
Suitable processes include:
(1) drum drying of principal ingredients, optionally followed by granulation or postdosing of additional ingredients;
(2) non-tower granulation of all ingredients in a high-speed mixer/granulator, for example, a Fukae (Trade Mark) FS series mixer, preferably with at least one surfactant in paste form so that the water in the surfactant paste can act as a binder;
(3) non-tower granulation in a high speed/moderate speed granulator combination, thin film flash drier/evaporator or fluid bed granulator.

Abstract: A detergent composition comprising:

Abstract: (c) the balance, if any, being other detergent ingredients wherein said composition comprises less than 35 wt %, preferably less than 25 wt %, more preferably less than 15 wt % of non-functional non-alkaline water soluble inorganic salt.

Unilever Home and Personal Care USA division of Conopco, Inc.


This invention relates generally to methods and apparatus for desorption and ionization of analytes for the purpose of subsequent scientific analysis by such methods, for example, as mass spectrometry or biosensors. More specifically, this invention relates to the field of mass spectrometry, especially to the type of matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry used to analyze macromolecules, such as proteins or biomolecules. Most specifically, this invention relates to the sample probe geometry, sample probe composition, and sample probe surface chemistries that enable the selective capture and desorption of analytes, including intact macromolecules, directly from the probe surface into the gas (vapor) phase without added chemical matrix.

1. Field of the Invention
This invention relates generally to methods and apparatus for desorption and ionization of analytes for the purpose of subsequent scientific analysis by such methods, for example, as mass spectrometry or biosensors. More specifically, this invention relates to the field of mass spectrometry, especially to; the type of matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry used to analyze macromolecules, such as proteins or biomolecules.
2. Description of the Prior Art
Generally, analysis by mass spectrometry involves the vaporization and ionization of a small sample of material, using a high energy source, such as a laser, including a laser beam. The material is vaporized from the surface of a probe tip by the laser beam, and in the process, some of the individual molecules are ionized by the gain of a proton. The positively charged ionized molecules are then accelerated through a short high voltage field and let fly into a high vacuum chamber, at the far end of which they strike a sensitive detector surface. Since the time of flight is a function of the mass of the ionized molecule, the elapsed time between ionization and impact can be used to determine the molecule""s mass which, in turn, can be used to identify the presence or absence of known molecules of specific mass.
All known prior art procedures which present proteins or other large biomolecules on a probe tip for laser desorption/ionization time-of-flight mass spectrometry rely on a crystalline solid mixture of the protein or other analyte molecule in a large excess of acidic matrix material deposited on the bare surface of a metallic probe tip. (The sample probe tip typically is metallic, either stainless steel, nickel plated material or platinum). Immobilizing the analyte in such a matrix was thought to be necessary in order to prevent the destruction of analyte molecules by the laser beam. The laser beam strikes the mixture on the probe tip and its energy is used to vaporize a small portion of the matrix material along with some of the embedded analyte molecules. Without the matrix the analyte molecules are easily fragmented by the laser energy, so that the mass, and identity, of the original macromolecule is very difficult to determine.
This prior art procedure has several limitations which have prevented its adaptation to automated protein or other macrobiological molecules analysis. First, in a very crude sample it is necessary to partially fractionate (or otherwise purify the sample as much as possible) to eliminate the presence of excessive extraneous materials in the matrix/analyte crystalline mixture. The presence of large quantities of components may depress the signal of the targeted analyte. Such purification is time-consuming and expensive and would be very difficult to do in an automated analyzer.
Second, while the amount of analyte material needed for analysis by the prior art method is not large (typically in a picomole range), in some circumstances, such as tests on pediatric patients, analyte fluids are available only in extremely small volumes (microliters) and may be needed for performing several different analyses. Therefore, even the small amount needed for preparation of the analyte/matrix crystalline mixture for a single analysis may be significant. Also, only a tiny fraction (a few thousandths or less) of analyte used in preparing the analyte/matrix mixture for use on the probe tip is actually consumed in the mass spectrometry analysis. Any improvement in the prior art procedure which made it possible to use much less analyte to conduct the test would be highly advantageous in many clinical areas.
Third, the analyte protein, or other macromolecule, used in preparing the analyte matrix for use on the probe tip is not suitable for any subsequent chemical tests or procedures because it is bound up in the matrix material. Also, all of the matrix material used to date is strongly acidic, so that it would affect many chemical reactions which might be attempted on the mixture in order to modify the analyte molecules for subsequent examination. Any improvement in the procedure which made it possible to conduct subsequent chemical modifications or reactions on the analyte molecules, without removing them from the matrix or the probe tip, would be of enormous benefit to researchers and clinicians.
Additional limitations in the prior art included problems with matrix use such as:
(1) formation of analyte-matrix complex (referred to as xe2x80x9cmatrix adductxe2x80x9d which interferes with the accuracy of analyte measurement;
(2) inability to wash away contaminants present in analyte or matrix (e.g., other proteins or salts);
(3) formation of analyte-salt ion adducts;
(4) less than optimum solubility of analyte in matrix;
(5) signal (molecular ion) suppression xe2x80x9cpoisoningxe2x80x9d due to simultaneous presence of multiple components; and
(6) selective analyte desorption/ionization.
There are a number of problems and limitations with the prior art methods. Prior investigators, including Karas and Hillenkamp have reported a variety of techniques for analyte detection using mass spectroscopy, but these techniques suffered because of inherent limitations in sensitivity and selectivity of the techniques, specifically including limitations in detection of analytes in low volume, undifferentiated samples. The xe2x80x9cHillenkamp-Karasxe2x80x9d articles that pertain to this field of invention are:
1. Hillenkamp, xe2x80x9cLaser Desorption Mass Spectrometry: Mechanisms, Techniques and Applicationsxe2x80x9d; Bordeaux Mass Spectrometry Conference Report, 1988, pages 354-362.
2. Karas and Hillenkamp, xe2x80x9cUltraviolet Laser Desorption of Proteins Up to 120,000 Daltonsxe2x80x9d, Bordeaux Mass Spectrometry Conference Report, 1988, pages 416, 417.
3. Karas and Hillenkamp, xe2x80x9cLaser Desorption Ionization of Proteins With Molecular Masses Exceeding 10,000 Daltonsxe2x80x9d, Anatical Chemistry, 60. 2299, July 1988.
4. Karas, Ingendoh, Bahr and Hillenkamp, xe2x80x9cUV-Laser Desorption/Ionization Mass Spectrometry of Femtomol Amounts of Large Proteinsxe2x80x9d, Biomed. Environ. Mass Spectrum (in press).
The use of laser beams in time-of-flight mass spectrometers is shown, for example, in U.S. Pat. Nos. 4,694,167; 4,686,366, 4,295,046, and 5,045,694, incorporated by reference.
The first successful molecular mass measurements of intact peptides and small proteins (only up to about 15 kDa) by any form of mass spectrometry were made by bombarding surfaces with high energy particles (plasma desorption and fast atom bombardment mass spectrometry); this breakthrough came in 1981 and 1982. Improvements came in 1985 and 1986, however, yield (signal intensities), sensitivity, precision, and mass accuracy remained relatively low. Higher molecular mass proteins (about 20 to 25 kDa) were not observed except on rare occasions; proteins representing average molecular weights (approximately 70 kDa) were not ever observed with these methods. Thus, evaluation of most proteins by mass spectrometry remains unrealized.
In 1988, Hillenkamp and his coworkers used UV laser desorption time-of-flight mass spectrometry and discovered that when proteins of relatively high molecular mass were deposited on the probe tip in the presence of a very large molar excess of an acidic, UV absorbing chemical matrix (nicotinic acid) they could be desorbed in the intact state. This new technique is called matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Note that laser desorption time-of-flight mass spectrometry (without the chemical matrix) had been around for some time, however, there was little or no success determining the molecular weights of large intact biopolymers such as proteins and nucleic acids because they were fragmented (destroyed) upon desorption. Thus, prior to the introduction of a chemical matrix, laser desorption mass spectrometry was essentially useless for the detection of specific changes in the mass of intact macromolecules (see below). Note that the random formation of matrix crystals and the random inclusion of analyte molecules in the solid solution is prior art.
The primary object of the invention is to provide improved methods, materials composition and apparatus for coupled adsorption, desorption and ionization of multiple or selected analytes into the gas (vapor) phase, preferably for use in conjunction with mass spectrometry of biomolecules and other macromolecules, as well as by means of analytic detection other than mass spectrometry. The invention incudes a flexible variety of options for presenting surfaces with attached energy absorbing molecules, defined reaction sites, and affinity reagents for the capture,e transfer, and/or the desorption of analytes before and after a sseries of chemical, physical, and/or enzymatic modifications performed in situ.
Another object is to provide such a method and apparatus for affinity-directed detection of analytes, including desorption and ionization of analytes in which the analyte is not dispersed in a matrix solution or crystalline structure but is presented within, on or above an attached surface of energy absorbing xe2x80x9cmatrixxe2x80x9d material through molecular recognition events, in a position where it is accessible and amenable to a wide variety of chemical, physical and biological modification or recognition reactions.
The probe surface with and without bonded energy absorbing molecules, referred to as the sample presenting surface can be composed of a variety of materials, including porous or nonporous materials, with the porous materials providing sponge-like, polymeric, high surface areas for optimized adsorption and presentation of analyte.
These surface materials can be substituted (at varying densities) with chemically bonded (covalent or noncovalent) affinity adsorption reagents and/or chemically bonded (i.e., immobilized) energy absorbing molecules (bound xe2x80x9cmatrixxe2x80x9d molecules). The geometry of the sample presenting surface can be varied (i.e., size, texture, flexibility, thickness, etc.) to suit the need (e.g., insertion into a living organism through spaces of predetermined sizes) of the experiment (assay).
Another object is to provide such a method and apparatus in which the analyte material is chemically bound or physically adhered to a substrate forming a probe tip or other sample presenting surface.
A further object is to, provide means for the modification of probe or sample presenting surfaces with energy-absorbing molecules to enable the successful desorption of analyte molecules without the addition of exogenous matrix molecules as in prior art.
A further object is to provide the appropriate density of energy-absorbing molecules bonded (covalently or noncovalently) in a variety of geometries such that mono layers and multiple layers of attached energy-absorbing molecules can be used to facilitate the desorption of analyte molecules of varying masses. The optimum ratio of adsorbed or bonded energy-absorbing molecules to analyte varies with the mass of the analyte to be detected. A further object is to modify the sample presenting surface with such energy-absorbing molecules where the composition of the probe or sample presenting surface is other than the metal or metallic surfaces as described in prior art. Separate from the chemical and/or physical modification of the probe surface with energy absorbing molecules is the modification of these surfaces with affinity reagents, both chemical and/or biological, for the specific purpose of capturing (adsorbing) specific analyte molecules or classes of analyte molecules for the subsequent preparation, modification, and successful desorption of said analyte molecules.
A further object is to provide all combinations of surfaces modified with energy-absorbing molecules and/or affinity-directed analyte capture devices to enable the selective and/or nonselective adsorption of analytes and the subsequent desorption either with or without requiring the subsequent addition of additional matrix molecules. It is important to note that the surfaces modified with affinity reagents for the capture of analytes are more useful than the underivitized sample surfaces described in prior art even when the deposition of energy-absorbing molecules (that is matrix) is as described in prior art. Because of the advantages in the ability to remove contaminating substances from the adsorbed analyte molecules and because of the ability to modify adsorbed analyte molecules without (or before) added matrix.
A further object is to provide such a method and apparatus in which the substrate forming the probe tip or other sample presenting surface is derivatized with one or more affinity reagents (a variety of densities and degrees of amplification) for selective bonding with predetermined analytes or classes of analytes.
A further object is to provide methods and apparatus for using probe tips having surfaces derivatized with affinity reagents and containing laser desorption matrix material (chemically bonded to surface or not) which may be used to isolate target analyte materials from undifferentiated biological samples such as blood, tears, urine, saliva, gastrointestinal fluids, spinal fluid, amniotic fluid, bone marrow, bacteria, viruses, cells in culture, biopsy tissue, plant tissue or fluids, insect tissue or fluids, etc.
Because of the new and preferred method for presentation and desorption of selected analytes, a further object is to use analyte detection methods other than the generic electron multipliers typically used in mass spectrometric devices. This would include but would not be limited to detection films/plates for the qualitative or quantitative evaluation of fluorescent or radio-labeled analytes or analyte molecule complexes.
A further object is to provide such a system in which the affinity reagent chemically bonds or biologically adheres to the target analyte or class of analytes.
A further object is to use existing and new solid phase affinity reagents (e.g., small diameter porous or nonporous beads of crosslineated polymer with attached molecular capture devices) designed for the (1) capture (adsorption of one or more analytes, (2) the preparation of these captured analytes (e.g., washing with H2O or other buffered or nonbuffered solutions to remove contaminants such as salts, multiple cycles of washing, such as with polar organic solvent, detergent-dissolving solvent, dilute acid, dilute base or urea), and (3) most importantly, the direct transfer of these captured and prepared analytes to the probe surface for subsequent analyte desorption (for detection, quantification and/or mass analysis).
A further object is to provide such a system in which the predetermined analytes are individual biomolecules or other macromolecules or combinations of adjoined molecules (i.e., complexes).
A still further object is to provide such a method and apparatus in which the matrix materials used are not strongly acidic, as in prior art matrices, but are chemically modified into the slightly acidic, neutral pH or strongly basic range of pH.
A further object is to provide such a system in which the matrix material has a pH above 6.0.
A still further object is to provide a method and apparatus for desorption and ionization of analytes in which unused portion of the analytes contained on the presenting surface remain chemically accessible, so that a series of chemical and/or enzymatic or other treatments (e.g., discovery of analyte-associated molecules by molecular recognition) of the analyte may be conducted on the probe tip or other presenting surface, in situ, followed by sequential analyses of the modified analyte by mass spectrometry. In one case (i.e., repetitive sequential analyses) the analyte is adsorbed to the sample presenting surface and can be treated (modified in situ after the excess free matrix is removed (i.e., washed away). Matrix can be added back before analysis by mass spectrometry. Using this procedure, an analyte can be repeatedly tested for a variety of components by removing one matrix, modifying the analyte sample, re-applying the same or different matrix, analyzing the sample, etc.
A further object is to provide a method and apparatus for the combined chemical and/or enzymatic modifications of target analytes for the purpose of elucidating primary, secondary, tertiary, or quaternary structure of the analyte and its components.
A still further object is to provide such a method and apparatus in which the probe tips or other sample presenting surfaces are; formed of a variety of materials, including electrically insulating materials (porous and nonporous), flexible or nonrigid materials, optically transparent materials (e.g., glass, including glass of varying densities, thicknesses, colors and with varying refractive indices), as well as less reactive, more biocompatible materials (e.g., biopolymers such as agarose, dextran, cellulose, starches, peptides, and fragments of proteins and of nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These surfaces can be chemically modified by the attachment of energy-absorbing molecules and/or affinity directed analyte capture molecules.
Another object is to provide a method and apparatus for desorption and ionization of analyte materials in which cations other than protons (H+) are utilized for ionization of analyte macromolecules.
Note that the laser or light source used to convey energy to the probe surface can employ a wavelength(s) that is(are) not fixed but can be varied according to the wavelength absorbed by the matrix (whether the matrix is added in the free form or is chemically bonded to the probe (sample presenting) surface). For this procedure, a variety of wavelengths (10 or more) defined by absorbance of matrix or energy absorbing surface can be utilized.
Another object is to provide such a method and apparatus in which the probe tips or other sample presenting surfaces used for laser desorption/ionization time-of-flight mass spectrometry are magnetized and in which the matrix, affinity directed absorption molecules and/or analyte materials are magnetically adhered to such magnetized surface.
A further object is a method and apparatus in which the matrix and/or analyte materials are adhered by any variety of chemical mechanisms to the sample presenting surface.
A further objective is to provide energy-absorbing molecules which have been incorporated into other chemical structures (e.g., chemical or biological polymers) for the deposition (covalent or noncovalent) onto the sample presenting surface in a way that enables repetitious analyte desorption events without interference with chemical and/or enzymatic modifications of the analyte molecule(s) performed in situ.
Another object is to provide sample presenting surfaces in a variety of sizes and configurations (up to 4xe2x80x3xc3x974xe2x80x3) with multiple (up to 10,000 or more) spots (including spots down to  less than 0.001 inch diameter) of affinity reagents arranged in predetermined arrays for the selective adsorption of numerous different analytes (e.g., clinical chemical marker proteins) to enable a wide spectrum sampling of the macromolecular composition of biological samples/fluids.
As shown more fully below, the present invention overcomes limitations and disabilities of the prior art by providing probe tips or sample plates whose surfaces have been derivatized with biospecific affinity reagents which will selectively bind specific groups or types of biomolecules or other analytes out of an undifferentiated sample (such as blood or urine). Appropriate selection of the affinity reagents used to derivatize the probe tip surface therefore makes possible the selection from the undifferentiated sample and binding to the probe tip of the specific types or groups of biological or other macromolecules under investigation, or subsequent examination (e.g., quantification and/or structure elucidation) by mass spectrometry. This has the advantage of achieving both the purification of the analyte sample previously required and the effect of concentrating the analyte. It reduces by a factor of 1,000 to 100,000 the amount of analyte needed for the mass spectrometry examination, since only the macromolecules which attach to the biospecific affinity reagents are removed from the analyte sample, and these can be sequestered on predetermined areas of the probe tips or sample plates that are even less than the laser spot size.
It also has been found that the probe tips used in the process of the invention need not be metal or metal-coated, as with prior art procedures. Research involved in the invention has involved glass and synthetic polymer surfaces such as polystyrene, polypropylene, polyethylene, polycarbonate and other polymers including biopolymers, for the probe tips which hate been covalently or noncovalently derivatized for immobilization of specific reagents that will direct the selective adsorption of specific analytes. These surfaces will include immobilized metal ions, immobilized proteins, peptides, enzymes, and inhibitor molecules, immobilized DNA and RNA, immobilized antibodies, immobilized reducing agents, immobilized carbohydrates and lectins, immobilized dyes and immobilized protein surface domains involved in molecular recognition (e.g., dimerization domains and subunits). Some of the chemical and surface structures are as yet unknown.
The preferred probe tip, or sample plate, for selective adsorption/presentation of sample for mass analysis are (1) stainless steel (or other metal) with a synthetic polymer coating (e.g., cross-linked dextran or agarose, nylon, polyethylene, polystyrene) suitable for covalent attachment of specific biomolecules or other nonbiological affinity reagents, (2) glass or ceramic, and/or (3) plastic (synthetic polymer). The chemical structures involved in the selective immobilization of affinity reagents to these probe surfaces will encompass the known variety of oxygen-dependent, carbon-dependent, sulfur-dependent, and/or nitrogen-dependent means of covalent or noncovalent immobilization. The methods and chemical reactions used in producing such surfaces derivatized with biospecific affinity reagents already are known by those skilled in the art. Two features of the invention, however, are (1) the specific size and localization of the derivatized surface with respect to the laser beam and (2) the affinity directed presentation of specific analyte molecules (e.g., macromolecule or biopolymer) at a defined surface density or local concentration required for the efficient detection by laser desorption/ionization time-of-flight mass spectrometry. This can be accomplished by arranging the affinity adsorption xe2x80x9cspotsxe2x80x9d (0.005 to 0.080 inch diameter) on the probe surface in a defined manner (400 to 1,000 spots could be placed on a surface about the size of a glass slide).
An additional discovery involves the fact that pH modified chemical matrices can be used on these surfaces to facilitate desorption/ionization without disruption of conditions necessary for subsequent sample modification. As discussed above, prior art matrix materials used for biomolecular mass spectrometry are acidic. The exact chemical structure of the pH-modified matrices still are unknown. However, by suitable neutralization of the matrix material, it can be made largely passive to subsequent chemical or enzymatic reactions carried out on the analyte molecules presented on the derivatized probe tip surface by the biospecific affinity reagents. This makes possible the carrying out of chemical reactions on the analyte molecules presented on the probe tips. Since only a small fraction of the analyte molecules are used in each desorption/mass spectrometer measurement, a number of sequential chemical and/or enzymatic modifications of the samples, in situ, on the probe tips, and subsequent analysis of the modified samples by mass spectrometry, can be carried out on the same probe tips in order to more accurately determine exactly what molecule is present, or other characteristics or information about the molecule, including its structure.
Finally, even when these matrix molecules are immobilized on a probe tip surface, the analyte deposited on such a surface can be desorbed with a laser beam. This circumvents the contamination of the analyte by the matrix molecules. As a particular feature of the invention, we have shown that some energy absorbing molecules that do not work as xe2x80x9cmatrixxe2x80x9d molecules when added to analytes as a solution of free molecules (as in prior art) do indeed work well to facilitate the desorption of intact analyte molecules after being immobilized.
It seems likely that these improvements in the procedure will enable bioanalytical and medical instrument manufacturers to develop a machine for the automated evaluation of a single protein sample deposited on a surface and modified with numerous chemical and/or enzymatic reactions performed in situ.

Method and apparatus for desorption and ionization of analytes


The present invention provides a hard gelatine capsule containing a pharmaceutical composition comprising cyclosporin A in a mixture with a surfactant of HLB value at least 10, substantially free of any oil and when a hydrophilic phase is present, the hydrophilic phase being a polyethylene glycol and/or a lower alkanol provided that any lower alkanol present is present in less than 12% of the total weight of the composition absent the hard gelatine capsule.

The present invention relates to novel pharmaceutical compositions comprising cyclosporin A, also known as ciclosporine, as active agent (hereinafter referred to as cyclosporin).
Hitherto few pharmaceutical compositions containing cyclosporin have been accepted for commercial use for humans. Thus in the USA only SANDIMMUNE and NEORAL (cyclosporin for microemulsion) have been approved.
These formulations are available in the form of a drink solution or a soft gelatine capsule. Such soft gelatine capsules require special manufacturing techniques.
The compositions of the present invention are compositions containing cyclosporin which meet the requirements for approval in the US or elsewhere, yet can be produced in a form administrable as a hard gelatine capsule. Such capsules are well known in the art and may be made and filled in conventional manner.
In one aspect to present invention provides an oral pharmaceutical composition comprising cyclosporin A in a mixture with (i) a surfactant of HLB value at least 10, and optionally (ii) a viscosity increasing agent and/or (iii) a hydrophilic phase, the hydrophilic phase being a polyethylene glycol and/or a lower alkanol provided that any lower alkanol present is present in less than 12%, preferably less than 10 or 8% of the total weight of the composition, the composition being adapted for filling into, and serving as a centre-fill for, a hard gelatine capsule, and being substantially free of any additional oil.
The present compositions are based on the use of very few components, e.g. a surfactant (including associated side products normally arising from its preparation), optionally a viscosity increasing agent (thickener) and if desired an additional hydrophilic phase (additional to that present in the surfactant) chosen from polyethylene glycol and/or a lower alkanol which said lower alkanol is present in an amount of less than 12%, e.g. 8% by weight of the composition.
Cyclosporin compositions which have been proposed before suffer from the disadvantage that they are not stable in hard gelatine capsules, e.g. over 2 to 3 years and have bioavailability or variability similar to SANDIMMUNE OR NEORAL. The present compositions have excellent stability. The capsules do not become brittle.
Preferably the composition contains few other excipients. This has the advantage of reducing bulk. Thus preferably less than 5%, preferably less than 2% or 1% of lipophilic moieties (oils) apart from those present in the surfactant, or hydrophilic moieties, e.g. alkanols such as ethanol or propylene glycol are present.
The compositions may contain polyethylene glycol. This may be a part of the surfactant for example if this is produced by polyethoxylation or added separately. This may be present from e.g. 1 to 40% of the formulation. Preferably the polyethylene glycol is liquid at 37xc2x0 C. e.g. having a M.W. 200 to 600 daltons.
The cyclosporin may be present in the usual dosage form for a cyclosporin formulation e.g. 25 mg; 50 mg; 100 mg per weight dosage form. The dosage form is e.g. a hard gelatine capsule as known in the art.
By the present invention there are provided novel cyclosporin galenic formulations, which meet or substantially reduce difficulties in cyclosporin, therapy hitherto encountered in the art. In particular it has been found that the compositions of the invention permit the preparation of solid, semi-solid and liquid compositions containing a cyclosporin in sufficiently high concentration to permit convenient oral administration, while at the same time achieving improved efficacy, e.g. in terms of bioavailability characteristics.
More particularly it has been found that compositions in accordance with the present invention enable effective cyclosporin dosaging with concomitant enhancement of resorption/bioavailability levels, as well as reduced variability in resorption/bioavailability levels achieved both for individual patients receiving cyclosporin therapy as well as between individuals. By application of the teachings of the present invention cyclosporin dosage forms are obtainable providing reduced variability in achieved cyclosporin blood/blood serum levels between dosages for individual patients as well as between individuals/individual patient groups. The invention thus enables reduction of cyclosporin dosage levels required to achieve effective therapy. In addition it permits closer standardisation as well as optimisation of on-going daily dosage requirements for individual subjects receiving cyclosporin therapy as well as for groups of patients undergoing equivalent therapy.
By closer standardisation of individual patient dosaging rate and blood/blood-serum level response, as well as dosaging and response parameters for patient groups, monitoring requirements may be reduced, thus substantially reducing the cost of therapy.
By reduction of required cyclosporin dosaging/standardisation of achieved bio-availability characteristics, the present invention also offers a means permitting reduction in the occurrence of undesirable side-effects, in particular nephrotoxic reaction, in patients undergoing cyclosporin therapy.
The present compositions are of a small volume, yet stable, thereby increasing patient compliance.
The surfactant is preferably approved by the FDA, e.g. a GRAS surfactant, e.g.
1.1 Polyethyloxylated castor oil, e.g. reaction products of natural or hydrogenated vegetable oils and ethylene glycol, i.e. polyoxyethylene glycolated natural or hydrogenated vegetable oils, for example polyoxyethylene glycolated natural or hydrogenated castor oils. Such products may be obtained in known manner, e.g. by reaction of a natural or hydrogenated castor oil or fractions thereof with ethylene oxide, e.g. in a molar ratio of from about 1:35 to about 1:60, with optional removal of free polyethyleneglycol components from the product, e.g. in accordance with the methods disclosed in German Auslegeschriften 1,182,388 and 1,518,819. Especially suitable are the various tensides available under the trade name Cremophor. Particularly suitable are the products Cremophor RH 40 having a saponification no. ca. 50-60, an acid no.= less than 1, an iodine no.= less than 1, a water content (Fischer)= less than 2%, an nD60=ca. 1,453-1,457 and an HLB=ca. 14-16; Cremophor RH 60 having a saponification no.=ca. 40-50, an acid No.= less than 1, an iodine no.= less than 1, a water content (Fischer)=ca. 4.5-5.5%, an nD25=ca. 1.453-1.457 and an HLB=ca. 15-17; and Cremophor EL having a molecular weight (by steam osmometry)=ca. 1630, a saponification no.=ca. 65-70, an acid no.=ca. 2, an iodine no.=ca. 28-32 and an nD25=ca. 1.471 (c.f. Fiedler loc. cit. pp. 326-327). Also suitable for use in this category are the various tensides available under the trade name Nikkol, e.g. Nikkol HCO-60. The said product Nikkol HCO-60 is a reaction product of hydrogenated castor oil and ethylene oxide exhibiting the following characteristics: Acid no.=ca. 0.3; Saponification no.=ca. 47.4; Hydroxy value=ca. 42.5; pH (5%)=ca. 4.6; Color APHA=ca. 40; m.p.=ca. 36.0 C; Freezing point=ca. 32.4 C; H2O content (%, KF)=ca. 0.03;
Such products contain a xe2x80x9chydrophilic portionxe2x80x9d of ca. 70 to 90% of fatty acid esters of glycerol polyethylene glycol, as well as fatty acid esters of polyethylene glycols and a hydrophilic portion of polyethylene glycol and glycerol ethoxylates. See for example Karl Mxc3xcller, Tenside, Year 3, Issue 2, p. 37-45.
Preferably the surfactant is a polyethoxylated hydrogenated castor oil Cremophor RH.
1.2 Polyoxyethylene-sorbitan-fatty acid esters (polysorbates) e.g. produced by co-polymerising ethylene oxide with fatty acid esters of a sorbitol and its anhydrides of e.g. mono- and tri-lauryl, palmityl, stearyl and oleyl esters e.g. of the type known and commercially available under the trade name Tween (c.f. Fiedler, loc. cit. pp. 1300-1304) including the products Tween
20 [polyoxyethylene(20)sorbitanmonolaurate],
40 [polyoxyethylene(20)sorbitanmonopalmitate],
60 [polyoxyethylene(20)sorbitanmonostearate],
80 [polyoxyethylene(20)sorbitanmonooleate],
65 [polyoxyethylene(20)sorbitantristearate],
85 [polyoxyethylene(20)sorbitantrioleate],
21 [polyoxyethylene(4)sorbitanmonolaurate],
61 [polyoxyethylene(4)sorbitanmonostearate], and
81 [polyoxyethylene(5)sorbitanmonooleate].
Especially preferred products of this class for use in the compositions of the invention are the above products Tween 40 and Tween 80;
1.3 Polyoxyethylene fatty acid esters, e.g. produced by reacting fatty acids with ethylene oxide, e.g. polyoxyl 40 stearate, for example polyoxyethylene stearic acid esters of the type known and commercially available under the trade name Myrj (c.f. Fiedler, loc. cit., p. 834) as well as polyoxyethylene fatty acid esters known and commercially available under the trade name Cetiol HE. (c.f. Fiedler, loc. cit., p. 284); an especially preferred product of this class for use in the compositions of the invention is the product Myrj 52 having a D25=ca. 1.1., m.p.=ca. 40-44 C, an HLB=ca. 16.9., an acid no.=ca. 0-1 and a saponification no.=ca. 25-35;
1.4 Polyethoxylated glyceryl fatty acid mono esters, e.g. of lauryl, stearic, oleyl, or isostearic acid, e.g. those obtainable under the name Tagat O or L.
1.5 Polyoxyethylene mono esters of a saturated C10 to C22, e.g. C18 substituted e.g. hydroxy fatty acid; e.g. 12 hydroxy stearic PEG acid, e.g. of PEG about e.g. 600-900 e.g. 660 daltons MW, e.g. SOLUTOL H515 from BASF, Ludwigshafen, Germany.
1.6 Polyoxyethylene-polyoxypropylene co-polymers, poloxamers, e.g. of the type known and commercially available under the trade names Pluronic and Emkalyx (c.f. Fiedler, loc. cit., pp. 956-958). An especially preferred product of this class for use in the compositions of the invention is the product Pluronic F68 (poloxamer 188).
1.7 Propylene glycol mono- and di-fatty acid esters such as propylene glycol dicaprylate, propylene glycol dilaurate, propylene glycol hydroxystearate, propylene glycol isostearate, propylene glycol laurate, propylene glycol ricinoleate, propylene glycol stearate and so forth (c.f. Fiedler, loc. cit., pp. 1013 et seq.). Especially preferred is propylene glycol caprylic-capric acid diester as known and commercially available under the trade name Miglyol 840 (c.f. Fiedler, loc. cit., p. 809). Miglyol 840 has a fatty acid content=C6 max. ca. 3%, C8 ca. 65-80%, C10 ca. 15-30%, C12 max. 3%. Acid no.=max. 0.1, iodine no.=ca. 320-340, iodine no.=max. 1.
Examples of ionic surfactants include:
2.1 Dioctylsuccinate, dioctylsodiumsulfosuccinate, di-[2-ethylhexyl]-succinate or sodium lauryl sulfate.
2.2 Phospholipids, in particular lecithins (c.f. Fiedler, loc. cit., pp. 731-733). Lecithins suitable for use in the compositions of the invention include, in particular, soya bean lecithins.
2.3 Bile salts, e.g. alkali metal salts, for example sodium taurocholate.
Examples of further lipophilic surfactants for use as surfactant component are, e.g.:
2.1 Trans-esterification products of natural vegetable oil triglycerides and polyalkylene polyols. Such trans-esterification products are known from the art and may be obtained e.g. in accordance with the general procedures described in U.S. Pat. No. 3,288,824. They include trans-esterification products of various natural (e.g. non-hydrogenated) vegetable oils for example, maize oil, kernel oil, almond oil, ground nut oil, olive oil and palm oil and mixtures thereof with polyethylene glycols, in particular polyethylene glycols having an average molecular weight of from 200 to 800. Preferred are products obtained by trans-esterification of 2 molar parts of a natural vegetable oil triglyceride with one molar part of polyethylene glycol (e.g. having an average molecular weight of from 200 to 800). Various forms of trans-esterification product of the class defined are known and commercially available under the trade name Labrafil [see Fiedler, loc. cit., 707]. Especially useful as components of the compositions of the invention are the products: Labrafil M 1944 CS, a trans-esterification product of kernel oil and polyethylene glycol having an acid no.=ca. 2, a saponification no. ca. 145-175 and an iodine no.=ca. 60-90; and Labrafil M 2130 CS, a trans-esterification product of a C12- to C18-glyceride and polyethylene glycol having a melting point=ca. 35-40 C., an acid no.= less than 2, a saponification no.=ca. 185-200 and an iodine no.= less than 3;
2.2 Mono-, di- and mono/di-glycerides, especially esterification products of caprylic or capric acid with glycerol. Preferred products of this class are e.g. those comprising or consisting mainly or essentially of caprylic/capric acid mono- and di-glycerides such as are commercially available under the trade name Imwitor (c.f. loc. cit., pp. 645). A particularly suitable product of this class for use in the compositions of the invention is the product Imwitor 742, which is the esterification product of a mixture of ca. 60 p.p.w. caprylic acid and ca. 40 p.p.w. capric acid with glycerol. Imwitor 742 is typically a yellowish crystalline mass, liquid at ca. 26 C; acid no.=max. 2; iodine no.=max. 1; saponification no.=ca. 235-275: % monoglycerides=ca. 40-50%; free glycerol=max. 2%; m.p.=ca. 24-26 C; unsaponifiables=0.3% max.; peroxide no.=max. 1;
2.3 Sorbitan fatty acid esters e.g. of the type known and commercially available under the trade name Span, for example including sorbitan-monolauryl, -monopalmityl, -monostearyl, -tristearyl, -monooleyl and -trioleyl estersxe2x80x94(c.f. Fiedler, loc. cit., pp. 1139-1140);
2.4 Pentaerythritol fatty acid esters and polyalkylene glycol ethers, for example pentaerythrite-dioleate, -distearate, -monolaurate, -polyglycol ether and -monostearate as well as pentaerythrite-fatty acid esters (c.f. Fiedler, loc. cit. pp. 923-924);
2.5 Monoglycerides, e.g. glycerol monooleate, glycerol monopalmitate and glycerol monostearate, for example as known and commercially available under the trade names Myvatex, Myvaplex and Myverol (c.f. Fiedler, loc. cit., pp. 836), and acetylated, e.g. mono-and di-acetylated monoglycerides, for example as known and commercially available under the trade name Myvacet (c.f. Fiedler, loc. cit., pp. 835);
2.6 Glycerol triacetate or (1,2,3)-triacetin (c.f. Fiedler, loc. cit., pp. 952); and
2.7 Sterols and derivatives thereof, for example cholesterols and derivatives thereof, in particular phytosterols, e.g. products comprising sitosterol, campesterol or stigmasterol, and ethylene oxide adducts thereof, for example soya sterols and derivatives thereof, such as known under the trade name Generol (c.f. Fiedler loc. cit., p.p. 554 and 555) in particular the products Generol 122, 122 E5, 122 E10, and 122 E25.
It is to be appreciated that surfactants may be complex mixtures containing side products or unreacted starting products involved in the preparation thereof made by e.g. polyoxyethylation may contain another side product, e.g. polyethylene glycol.
The compositions of the invention may also comprise a thickening agent (also referred to as a viscosity increasing agent).
Suitable thickening agents may be of those known and employed in the art, including e.g. pharmaceutically acceptable polymeric materials and inorganic thickening agents which enable the compositions to be filled easily and resists leakage, e.g. thixotropic agents. These should also have the property of dissolving quickly (e.g. within 5 minutes) in the stomach juices or water or at pH 1 to 2, for example, of the following types:
3.1 Water soluble tocopheryl polyethylene glycol succinic acid esters (TPGS), e.g. with a polymerisation number ca 1000, e.g. available from Eastman Fine Chemicals Kingsport, Tex., USA.
3.2 Water soluble celluloses and cellulose derivatives including; alkyl celluloses, e.g. methyl-, ethyl- and propyl-celluloses; hydroxyalkyl-celluloses, e.g. hydroxypropyl-celluloses and hydroxypropylalkyl-celluloses such as hydroxypropyl-methyl-celluloses; acylated celluloses, e.g. cellulose-acetates, cellulose-acetatephthallates, cellulose-acetatesuccinates and hydroxypropylmethyl-cellulose phthallates; and salts thereof such as sodium-carboxymethyl-celluloses. Examples of such products suitable for use in accordance with the present invention are those known and commercially available, e.g. under the trade names Klucel and Methocel (c.f. Fiedler, loc. cit., pp. 688 and 790),
3.3 Water soluble polyvinylpyrrolidones, including for example poly-N-vinylpyrrolidones and vinylpyrrolidone co-polymers such as vinylpyrrolidone-vinylacetate co-polymers, especially of low molecular weight. Examples of such compounds suitable for use in accordance with the present invention are those known and commercially available, e.g. under the trade name Kollidon (or, in the USA, Povidone) (c.f. Fiedler, loc. cit., pp. 694-696), in particular the products Kollidon 30 and 90;
3.4 Low amounts of inorganic thickening agents such as atapulgite, bentonite and silicates including hydrophilic silicon dioxide products, e.g. alkylated (for example methylated) silica gels, in particular colloidal silicon dioxide products as known and commercially available under the trade name Aerosil [c.f. Handbook of Pharmaceutical Excipients, loc. cit., p.p. 253-256] in particular the products Aerosil 130, 200, 300, 380, O, OX 50, TT 600, MOX 80, MOX 170, LK 84 and the methylated Aerosil R 972.
The compositions may also include one or more further ingredients e.g. in an amount of from 0.1 to 5%, in particular anti-oxidants [e.g. ascorbyl palmitate, butyl hydroxy anisole (BHA), butyl hydroxy toluene (BHT) and tocopherols, e.g. -tocopherol (vitamin E)], flavouring agents and so forth. Use of an anti-oxidant, in particular a tocopherol, is particularly advantageous.
The relative proportion of ingredients in the compositions of the invention will, of course, vary considerably depending on the particular type of composition concerned. Determination of workable proportions in any particular instance will generally be within the capability of the man skilled on the art. All indicated proportions and relative weight ranges described below are accordingly to be understood as being indicative of preferred or individually inventive teachings only and not as not limiting the invention in its broadest aspect.
a) The cyclosporin will generally be present in an amount of from 5 to 30%, suitably from about 10 to about 25% by weight based on the total weight of the composition absent the hard gelatine capsule.
b) Any polyethylene glycol amount when present will generally be present in an amount of from about 15% to about 30%, by weight based on the total weight of the composition absent the hard gelatine capsule;
c) Any further excipient apart from the surfactant and as thickening agent is preferably present from an amount from 0.1% to 5%, by weight based on the total weight of the composition absent the hard gelatine capsule.
The compositions above may additionally include a thickening agent, though, as previously indicated, this will generally be less preferred. The amount of thickening agent present may vary e.g. depending on the required consistency of the end product, e.g. whether it is to be in a thickened flowable form, for example for filling into a capsule. The amount will of course also depend on the nature of the thickening agent chosen. In general the thickeners components (4), when present will be present in an amount of up to about 25% by weight based on the total weight of the composition, more suitably in an amount of up to about 15 or 20% by weight, e.g. in an amount of from 0.5 or 5 up to 15 or 20% by weight based on the total weight of the composition.
The compositions may also include further additives or ingredients, e.g. as hereinbefore described. In particular they may comprise antioxidants, e.g. in an amount of up to about 0.5 or 1% by weight based on the total weight of the composition, and sweetening or flavouring agents, e.g. in an amount of up to about 2.5 or 5% by weight based on the total weight of the composition.
Preferably no other excipients are present. Thus the volume may be kept low and the composition may be filled into a capsule size, 1, 2 or 3.
The compositions have been found to exhibit especially advantageous properties when administered orally, e.g. in terms of both the consistency and high level of bioavailability achieved as defined in standard tests in humans or e.g. beagle dogs. In particular, and in contrast with other galenic systems, e.g. as known from the art, it has been found that such compositions are compatible with tenside materials, e.g. bile salts, present in the gastrointestinal tract. That is, they are fully dispersible in aqueous systems comprising such natural tensides and are thus capable of providing microemulsion systems in situ which are stable and do not exhibit precipitation or other disruption of fine particulate structure. Function of such systems on oral administration remains independent of and/or unimpaired by the relative presence or absence of bile salts at any particular time or for any given individual. Such compositions accordingly represent an especially preferred embodiment of the invention. The bioavailability characteristics may be observed in standard clinical trials or in dogs using standard radioimmunoassays for cyclosporins. Preferred capsules have a short Tmax. Preferably the compositions form, on dilution with water, micellar solutions, in which one may be able to detect droplets of, e.g. from 10 to 150 nm in diameter.
The compositions above will preferably be compounded in orally administerable hard gelatine capsule shells to be unit dosage forms. Where the compositions are in unit dosage form, each unit dosage will suitably contain between about 5 or 10 and about 200 mg cyclosporin, more suitably between about 15 or 25 and about 150 mg, e.g. 25, 50 or 100 mg cyclosporin. Thus unit dosage forms in accordance with the invention, suitable for administration 1xc3x97, 2xc3x97 or 3xc3x97 up to 5xc3x97 daily (e.g. depending on the particular purpose of therapy, the phase of therapy etc . . . ) will appropriately comprise e.g. about 25 mg, about 50 mg or about 100 mg cyclosporin per unit dosage.
Further details of the excipients are given in Fiedler.
The following non-limitative Examples illustrate the invention.

Oil-free pharmaceutical compositions containing cyclosporin A


A framed PV module rooftop and ballasted ground mount racking system by which the mounting structure also functions as a wire management system and operates as an electrical raceway that includes a plurality of North-South mounting structures, oriented in a North-South direction, each of the North-South mounting structures including a North-South open-air channel for receiving wires, and each of the North-South mounting structures configured for supporting a solar module at a selected tilt angle; a plurality of East-West mounting structures, oriented in an East-West direction, each of the East-West mounting structures including an East-West open-air channel for receiving wires, wherein the coupling of said North-South mounting structures to said East-West mounting structures forms a continuous, open-air, electrical raceway with intersections within an array for module connectivity.

Many racking systems for mounting or supporting photo-voltaic (PV) solar panels or modules on roofs or on the ground are commercially available. PV systems are expected to have a 25-year lifespan, and array wire management solutions should match this expectancy. However, designers and installers face persistent challenges to managing free-air conductors within the PV array.
Implementing a safe, reliable and Code-compliant wire management system for exposed PV conductors is not an easy task. PV module leads can be too long or sometimes too short, requiring site-specific solutions for supporting and interconnecting the cables. Equipment grounding, PV source-circuit and inverter-output conductors from micro-inverters and alternate current (AC) modules must also be managed to ensure protection from potential damage. Within the PV array, these various conductors are often secured to module frames, support rails and other racking system components. While securing array wiring seems as though it should be a straightforward exercise, the reality is that integrators continue to face numerous challenges.
UV-rated plastic wire ties provide a flexible, convenient and low-cost solution for bundling and fastening array conductors, but they are not a permanent answer and require regular inspection and replacement. Stainless steel cable clips work well for attaching conductors to the module frame, but this solution is often limited to supporting only one or two conductors at a time, and not all modules have frames. Structural channels or integrated cable management solutions within the module support rails assist with protecting and routing conductors, yet precautions must be taken where conductors enter and exit the channels to protect them from sharp edges that may compromise their insulation. In addition, module rails tend to run either east-west or north-south, but not both, so the rails may not provide support where conductors run perpendicular to the particular rail alignment.
Unfortunately, the PV industry lacks consistent, standardized wire management solutions for free-air conductors within the array. Coupled with the harsh environmental conditions that PV array conductors are exposed to—UV radiation, high winds and extreme ambient temperatures, and ice, snow and other debris—wire management continues to be the Achilles heel of the majority of PV installations.
The majority of PV systems have integrated leads, or wires, used for making electrical connections between modules. The module leads and the associated homerun conductors are often installed in free air within the PV array—meaning they are not in conduit. These exposed circuits contain potentially lethal voltages and currents that could electrocute someone, or cause a fire, in a faulted condition.
Although many racking systems are available, it has been found that the systems do not provide labor predictability, or provide predictable results based on structural needs or on-site requirements.
As a result, there remains a need for wire management solutions that can endure the extreme environments in which PV arrays are installed while ensuring the systems are safe and reliable.

Wire management racking system for photovoltaic solar modules


The invention concerns a recombinant DNA vector characterized in that it is capable of directing the expression an/or transcription of a selected nucleotide sequence in the cells of the central nervous system and in that it comprises (i) at least part of the genome of an adenovirus, including the regions required for that adenovirus to penetrate into the cells normally infectable by that adenovirus and (ii) being inserted into said part of genome of an adenovirus under the control of a promoter, either present or also inserted into said genome part and operative in said cells. This recombinant vector finds particular use in the treatment of diseases of the central nervous system, also in gene therapy.

To transfer safely and efficiently therapeutic DNA into the central nervous system, is a formidable challenge in the development of active therapies in brain diseases.
Preliminary investigations have been carried out with a number of vectors, more particularly retroviral vectors and herpes simplex derived vectors. However the usefulness of such gene transfer vehicles has, to date, been limited.
In most cases, retroviral vectors are not useful because they are unable to infect postmitotic cells, including most neural cells (1). Herpes simplex derived vectors infect neural cells but problems of pathogenicity and of stability of gene expression remain unsolved (2,3). In addition Herpes simplex derived vectors have so far proven to have but limited efficacy of expression. In a most recent article (4) the authors refer to the short-term expression reported earlier of HSV-1 derived vectors, because most of the promoters used had only been active during the acute phase of viral infection (less than 10 days post-infection). They disclosed the expression of a b-glucuronidase gene in a cell of the central nervous system under the control of the LAT promoter normally associated with the latency-associated-transcript (LAT) sequence of the virus. But the authors also report that, even though their experiments demonstrated the feasibility of using the LAT promoter for long-term expression of foreign genes in cells of the central nervous system to correct a genetic enzymatic deficiency in infected cells, too few cells had been corrected to alter the disease phenotype. Consequently their vector system needed to be improved to correct sufficient cells for obtaining a clinically significant effect.
The invention aims at obviating such difficulties and at providing most effective vector systems capable of delivering foreign genes and, where appropriate, their transcription products or expression products directly to cells of the central nervous system, particularly to terminally differentiated cells, incapable of proliferation. A more particular object of the invention is also to allow for the wide spreading of such vector systems throughout the neural tissue to be infected, yet while remaining substantially confined thereto.
Still another object of the invention is to produce such vector systems which are sufficiently safe to allow for a study and regulation in vitro of cloned genes in such cells or in test animals, and for therapy, in man or animal, involving the in situ production of a selected expression product, including gene therapy.
The invention is based on the recognition that adenovirus-derived vectors, particularly non-replicative adenovirus vectors, are capable of fulfilling these aims, both in vitro and in vivo. They provide powerful delivery systems of genes into the cells of the central nervous system, more particularly brain cells. They are characterized by a degree of infectivity of sufficient magnitude to allow for the infection of considerable populations of cells The biological experiments disclosed hereafter demonstrate the capability of adenovirus derived vectors (or adenoviral vectors) of efficiently infecting nerve cells, particularly neurons, both in vitro and in vivo.
Thus the invention provides a process for the production of a recombinant vector useful in a method comprising causing the transcription product or the expression product of a nucleotide sequence coding for a selected polypeptide to be targeted or produced in cells of the central nervous system, e.g. brain cells, particularly neural, glial or ependymal cells, wherein said recombinant vector is an adenoviral vector which comprises at least part of the genome of an adenovirus including those regions of that genome which provide the genetic information required by that adenovirus to penetrate into cells normally infectable by it, said nucleotide sequence being inserted in said genome part, under the control of a promoter either present or also inserted within said adenoviral vector, and said promoter being operative in said cells.
Thus the invention is more particularly related to the use of an adenovirus-derived vector for the expression of a selected nucleotide in the cells of the central nervous system.
The invention also provides a recombinant DNA vector characterized in that it is capable of directing the expression and/or transcription of a selected nucleotide sequence in the cells of the central nervous system and in that it comprises (i) at least part of the genome of an adenovirus, including the regions required for that adenovirus to penetrate into the cells of the central nervous system, and (ii) said selected nucleotide sequence under the control of a promoter operative in said cells.
The powerful capability of adenoviral vectors of transferring gene fragments in vivo into quiescent neural cells is illustrated by the experiments reported hereafter, which were carried out with an adenovirus vector carrying the E. Coli lac Z gene or the human tyrosine hydroxylase gene, in neural cells of adult rats. A large number of neural cells (including neurons, astrocytes, microglia and ependymal cells) expressed these transgenes at least 60 days after inoculation of various brain areas. Injecting up to 3xc3x97105 pfu in 10 xcexcl did not result in any detectable cytopathic effects, which were only observed for the highest titers of infection ( greater than 107 pfu/10 xcexcl) and were most likely associated with a massive endocytosis of viral particles in neural cells close to the injection site.
Moreover the genomes of adenoviruses can be manipulated to accommodate foreign genes of 7.5 kb in length or more. It has a large host range, a low pathogenicity in man, and high titers of the virus can be obtained (5).
It will be readily appreciated that these results strongly support the presumption that adenovirus offers, as a vector, a new and remarkable tool to genetically modify quiescent cells of the nervous system, because of its great efficacy of infection, long term expression, wide host range and low toxicity. Thus, adenovirus should be instrumental in the study of the function of cloned gene products in their physiological and anatomical context. The ability to infect the hippocampus (as this will be shown later) is of great interest to study integrated phenomena such as long-term potentiation, in animals.
Moreover the adenovirus clearly appears as an efficient means to transfer foreign genes into the brain with a therapeutic goal. Adenovirus vectors have great potential for gene therapy of nervous system diseases, such as the local delivery of growth factors or neurotransmitters for degenerative diseases and, more generally, to replace defective genes in appropriate cells. Relatively low titers of adenovirus vectors can efficiently transfer foreign genes into a significant number of brain cells without triggerring pathological effects. Subject to optimization of the doses of recombinant adenoviral vectors containing a foreign gene to be delivered into brain cells, they open new avenues in the treatment of many genetic and acquired neurological diseases, consequently, an alternative to drug treatment or brain transplantation of fetal tissues.
Adenovires, particularly adenoviruses of type 2 or 5 (Ad2 or Ad5) are particularly preferred. They are relatively stable, can be cultured easily and rapidly (viral cycle of about 30 hours) and provide high titers: up to 104-105 plaque forming units (p.f.u.) per infected cell. They are not oncogenic. The complete sequence of their viral genome has been established (6) and its molecular biology has been studied extensively. Finally several mutants, particularly deletion mutants, have been obtained, which makes it possible to insert fragments of large size therein (7).
Preferably the recombinant vectors for use in this invention are defective adenoviruses, whose genomes no longer contain those nucleotides sequences required for the virus replication in cells (other than brain cells) normally infectable by it. More particularly, they are free of the E1 region, including the early E1a region which activates the other early transcription units of the virus required for its replication as well as the region E1b involved in the establishment of a fully transformed phenotype, in the protection of DNA sequences during viral infection and required for the normal progression of viral events late in infection.
Preferably too, they are devoid of the E3 region which is involved in cellular immunity in vivo and is totally dispensible for growth in vitro.
Preferably however the recombinant vector for use in that invention comprises all of the sequences that are essential for the adenovirus encapsidation.
The promoter controlling the sequence coding for the selected polypeptide to be targeted or produced in the cells of the central nervous system, can either be endogenous or exogenous with respect to the adenoviral parts of the recombinant vector.
A preferred homologous promoter is any one that is likely to be recognized by the polymerases, practically RNA polymerase II, of human or animal cells infected by such adenoviruses. A particularly preferred endogenous promoter is the major late strong promoter (MLP) of the human adenovirus of type 2 (Levrero et al., 1991) (8). Another promoter that may be used consists of the early promoter of the E1a region of the adenovirus. In that last instance, a preferred defective adenovirus is devoid of its 5xe2x80x2 region normally upstream for that early promoter. In that last instance, the nucleotide sequence sought to be introduced in the neural cells is substituted for the E1A region.
The endogenous promoters of the adenoviruses can also be replaced by other ubiquitary promoters of heterologous or exogenous origin, e.g.:
a promoter contained in the LTR (Long Terminal Repeat) of the Rous Sarcome Virus (RSV) or the LAT promoter referred to above,
a promoter of the IE gene of Cytomegalovirus (CMV),
inducible MMTV promoters (originating from the mouse mammary tumor virus) or metallothionine promoters.
Other promoters can be used too. Particularly, neural or glial promoters will be preferred, particularly in instances where the nucleotide sequence inserted in the adenoviral vector is to be targeted more specifically on more specific classes of neural cells. Reference is for instance made to the following promoters, particularly those involved in the genes coding for neurotransmitter synthesizing enzymes:
TH (tyrosine hydroxylase)
CHAT (choline acetyl transferase)
TPH (tryptophane hydroxylase)
GFAP (glial fibrillary acidic protein) enolase g (neuronal protein marker) aldolase C.
The invention relates also to a process for making such recombinant vectors, which process comprises inserting the nucleotide sequence whose expression is sought in the starting vectors and the transformation of infectable cells with said vectors. Where the vector is a full live virus, the recombinant viruses are then recovered from the cell culture medium. Where the recombinant vector is a defective virus, a preferred process then comprises the transformation of a transformable eucaryotic cell line (preferably of human or animal origin) which itself comprises a distinct nucleotide sequence capable of complementing the part of the adenovirus genome that is essential for its replication and which is not present in said vector, whereby that complementation sequence is preferably incorporated into the genome of said cell line.
By way of preferred examples of such cell lines, one should mention the so called xe2x80x9c293 cell linexe2x80x9d derived from human embryonary kidney which contains, integrated in its genome, the first 11% of the 5xe2x80x2 region of an Ad5 virus genome. That portion of the Ad5 genome enables recombinant viruses defective in that region, because of a deletion of part that region, to be appropriately complemented. Such a process for the production of defective viruses has been described more particularly in European patent application nxc2x0 EP 185573, filed on Nov. 20, 1985.
After transformation of such cell lines, the defective recombinant viruses are multiplied, recovered and purified.
Needless to say that the same process may be applicable to the production of other defective adenoviruses as a result of a deletion in a region other than in the 5xe2x80x2 region referred to hereabove, it being then understood that the cell lines used in such production should then include in their own genome the sequence deleted form the adenoviral genome to thereby allow for the complementation of such defective adenoviruses.
The invention provides thus for the first time a serious alternative to injection in the brain area of cells, e.g. embryonic cells carrying the relevant genetic information, in gene therapy aiming at correcting metabolic deficiencies or defects in the targeted cells, particularly post-mitotic neurons. This is the consequence of the important infective power of adenoviruses, also retained by the corresponding defective adenovirus-derived vectors, of their capacity of spreading throughout the targeted neural or nerve tissue while also remaining substantially confined within the selected tissue, if specifically injected thereinto, as well as of the long-term transcription, and in most instances, expression of the nucleotide sequence carried into the nerve tissue by such adenoviral vectors.
The nucleotide sequence whose introduction in the cells of the central nervous system may be sought, may consist of any sequence capable of providing molecules interacting with the metabolism of such cells. Such molecules may consist of selected anti-sense RNAs, or anti-sense oligoribonucleotide, capable of interacting with defective messenger RNAs whose further processing, responsible for corresponding diseases, ought to be blocked, e.g. in a number of neuro-psychiatric diseases, epilepsy, etc . . . This methodology appears of particular interest in the treatment of the Alzheimer""s disease. The anti-sense method could be used, by way of example only, for inducing a blockade of the b-amyloid precursor, for preventing an accumulation of the b-amyloid peptide in the senile plaques. Alternatively use could be made of anti-sense oligonucleotide capable of inhibiting the expression of enzymes involved in the abnormal phosphorylation of proteins, e.g. the TAU protein involved in Alzheimer""s disease. Alternatively again the nucleotide sequence is one which could sequester specific binding proteins, themselves normally involved in the processing of the DNA or RNA sequence whose transcription or expression is sought to be inhibited.
The nucleotide sequence whose introduction in the cells of the central nervous system may be sought, may also code for an expression product having a biological property. Said expression product may for example be capable (1) of compensating a corresponding defective natural polypeptide containing product encoded by a defective corresponding nucleotide sequence, or (2) of compensating the lack of endogenous production of the natural endogenous corresponding polypeptide containing product in said targeted cells, or (3) of introducing new therapeutic activities in the infected cells. Examples of such defective polypeptide-containing products may consist of neurotransmitter-synthesizing enzymes and growth factors. For example in the case of Parkinson""s disease (which is characterized by a vulnerability of dopaminergic cells) one could envisage producing locally DOPA or dopamine by expressing the cDNA encoding tyrosine hydroxylase, or a growth factor such as BDNF (brain derived neurotrophic factor) which could favor the survival of dopaminergic neurons. Likewise for Alzheimer""s disease, where one of the missing neurotransmitters is acetylcholine which is synthesized by choline acetyl transferase. Moreover NGF (nerve growth factor) could prevent degeneration of cholinergic neurons.
Another potentially useful factor to express is CNTF (ciliary neurotrophic factor) which could prevent neuron death. But CNTF may also have interesting effect in the brain, e.g. for the blocking of the destruction (seemingly induced in diabetes affected patients) of peripheral nerves. Other trophic factor whose expression may be sought consist, by way of examples of IGF, GMF, aFGF, bFGF, NT3 and NT5.
In a general manner growth factors could be caused to be produced in neuronal cells of patients affected with neuropathies, strokes, spinal cord injury, amyotrophic lateral sclerosis, Huntington""s chorea, Alzheimer""s and Parkinson""s diseases, cerebral palsy. Epilepsia may be, among other possibilities, treated by a local production, in the central nervous system, of the neurotransmitter GABA, as a result of the expression of the glutamic acid decarboxylase.
The invention is also of particular interest for the preparation of compositions for use in the treatment of hereditary diseases affecting the defective or deficient product of the mutant gene: lysosomal enzymes in lysosomal disses (e.g. hexosaminidases in Tay Sachs and Sandhoff diseases, arylsulfatase in metachromatic leucodystrophy, glucocerebrosidase in Gaucher""s disease, b-glucursoronidase in mucopolysaccharidosis, HGPRT in Lesh Nyhan disease, etc . . . ). Hereditary progressive neuron degenerations could be treated by transfer of the normal disease gene via adenoviral vectors, or, as discussed above, by induction of a local production of growth factors. For instance, it has been shown, that production of CNTF could slow down progressive motoneuronal degeneration (pmm) of mice (Sendtner et al, Nature 1992; 358: 502-504), the same being observed with aFGF on photoreceptor degeneration in inherited retinal dystrophy in rat (Faktorovich et al, Nature 1990; 347:83-86). Acquired spinal cord diseases like the frequent and constantly lethal Amyotrophic Lateral Sclerosis (ALS) could perhaps benefit from similar local production of CNTF that has been proved to protect motoneurons.
Inherited dysmyelinating diseases could also be improved by adenovirus-mediated gene transfer into myelin-synthesizing cells.
Finally, some other types of potential therapeutic agents could be locally produced into the CNS, for instance enkephalins to attenuate rebel pains, for instance in cancerous patients.
The invention also relates to the pharmaceutical compositions consisting of the recombinant adenoviral vectors containing the nucleotide sequences as defined above, in association with a pharmaceutical carrier suitable for the administration route to be selected, e.g. direct in situ injection of the viral suspensions obtained in the relevant neural tissue (or though far less preferred, through a general route, e.g. intravenous route, particularly when the adenoviral vector also contains a promoter selectively operative in determined nerve tissue or cells).
The invention is not limited to the therapeutical uses contemplated hereabove, of the adenoviral vectors. The latter can also, owing to their high infectivity, be used either in in vitro assays on determined populations of neural cells, e.g. for the sake of studying the capacity of a promoter (then coupled to a suitable xe2x80x9cmarkerxe2x80x9d, e.g. b-gal) of being recognized by the polymerases of said neural cells. Alternatively such adenoviral vectors can also be used for the detection or the evaluation of the interaction recombinant virus (that expressed by the nucleotide sequence under the control of a promoter operative in such cells) with a given population of neural cells or a more complex nerve tissue. For instance that evaluation or detection may aim at localizing those cells of the more complex tissue which carry a receptor for the virus. The detection can make use of any appropriate classical labeled method, e.g. use of labeled antibodies to detect expression products of the nucleotide sequence assayed. The prospects of these evaluations may be considerable in the field of neuroanatomy.
The recombinant vector of this invention is also useful in a method comprising causing the transcription product or expression product of a nucleotide sequence coding for a selected polypeptide to be targeted or produced in cells of the central nervous system, e.g. brain and spinal cord cells, particularly neural, glial or ependymal cells, of an animal and detecting the resulting physiological or behavioral modification induced in said animal by said transcription product or said expression product.
For instance in the case of a lesion of the septohippocampic track which depletes the hippocampus in acetylcholine, it would be possible to study the effects of the transfer of a gene coding for the choline acetyl transferase (CHAT) in the hippocampus. The introduction of that gene in the target core of cholinergic fibers (cut by the lesion) could elicit a re-increase of the amount of the available acetyl choline and, consequently, correct the deficit. This deficit can be evaluated by behavioral tests of memorization. For instance mice thrawn in a xe2x80x9cswimming poolxe2x80x9d can learn to find again a platform which enables them to escape to water (Morris swimming pool). However the animals whose septo-hippocampic track has been deteriorated are very handicapped in that operation. The detection of an increase of the acetyl choline produced as a result of the gene expression would then be appreciated by the ease with which the animals would be able again to find the platform.
According to another example, the invention would enable the analysis of the degree of compensation of troubles of the motor behavior produced by the denervation of the striatum, particularly in the event of a lesion of the dopaminergic cores of the mesencephale (substance nigra). The introduction of the gene coding for tyrosine hydroxylase (TH) in the striatum could correct that deficit The compensation can be evaluated by the study of the behavior of rats in the rotation test: animals wounded on one side only rotate in a repetitive fashion (over ten rotations per minute) when they receive an injection of apomorphine. The production of dopamine (linked to the introduction of the TH gene) could be appreciated in that behavioral test.
Still according to another example, the invention allows for the electrophysiological and behavioral study in other cases. For instance, the neurones of the dorsal horn of the spinal cord which transmit information bound to nociceptive stimulation (pain channels) are sensitive to morphine which causes their activity to be decreased. The introduction of a gene coding for an endomorphine in the spinal cord could provoke a secretion of this substance which, like morphine, would act of these cells. Such an action could be evaluated by behavioral tests (threshold of reaction of animals to nociceptive stimulation) as well as by electrophysiological studies on the neurones themselves of the spinal cord. Recording of the activity of these cells would enable one to appreciate the existence of a modulation induced by the expression of the transferred gene.
The possibilities afforded by the invention will be further illustrated, yet in a non-limitative manner by the description of a number of assays, which are in part supported by figures appended to the present description. Particularly, the Ad.RSVbgal appears as an appealing means to analyze neuronal and glialmorphology in specific areas of the brain by providing Golgi-like staining of cells at the injection site. Filing up of the axons by the enzyme may additionally provide ways to analyze projections of discrete neuronal populations. Conversely uptake of the virus by the terminals and subsequent retrograde transport to the neuronal cell body allows tracing of sets of afferents to a specific brain area.
One major advantage of this technique for neuroanatomy is the easy combination of the X-gal stain with all sorts of other labeling techniques, in particular immunocytochemistry. The use of vectors in which the nls sequence would be omitted might improve the efficacy of such a technique.
Similarly that adenoviral vector provides a basis for the study of the action of other promoters substituted for the RSV LTR promoter, with respect to different neural cell populations.

Assays for promoter operability in central nervous system cells